GSM7011521: D15 EB, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
EBs were dissociated in 0.1% collagenase B (Roche Diagnostics, Laval, QC, Canada) for 1 hour at 37°C. They were then treated with 0.25% trypsin for 3 minutes and digested by gentle mechanical shaking. Mice were killed at week 20, and the femurs and tibias were removed. Bone marrow was flushed, single-cell suspensions were prepared by gently pipeting up and down several times followed by  rinsing and filtration on 40 μm cell strainers (Nylon mesh). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.